Changes in host cell membrane activities in response to adhesion of Neisseria gonorrhoeae

Physiological changes in host cell model membranes (intact human erythrocytes and ghosts) as a consequence of bacterial adhesion were studied with special reference to Neisseria gonorrhoeae. Membrane activities examined were transport of K+, Cl- ions, pyruvate kinase, Na-K-dependent ATPase, and cAMP. We found that K+ and Cl- transport were affected, more so in membranes with attached pilated (P+) organisms than in those with apilated (P-) isogenic strains. In N. gonorrhoeae and in several other species of gram-negative bacteria studied, hemagglutination titres were directly correlated with effects on anion transport, suggesting that perturbations in anion transport are an immediate result of adhesion. Of three P+ gonococcus strains tested, two depressed Na-K-ATPase activity in the membrane, indicating a possible effect on the Na-K pump. Pyruvate kinase activity associated with the membrane appeared to be stimulated by attached gonococci, again by P+ strains to higher levels than P- organisms. Clearly, some enzyme properties of host membranes are intrinsically affected by bacterial adhesion. Human polymorphonuclear neutrophils were also investigated, and with some exceptions, changes observed in leukocyte enzyme activities tended to parallel those in erythrocytes. Since hypochlorous acid production is considered to be an important microbicidal mechanism in neutrophils, interference with Cl- transport could jeopardize their role in host defense.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Apolipoprotein B mediates the capacity of low density lipoprotein to suppress neutrophil stimulation by particulates

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL). Liposomes containing gamma-palmitoyl-beta-oleoylphosphatidylcholine (PC) with unesterified cholesterol (PC:cholesterol = 2:1), PC and sphingomyelin (PC:sphingomyelin = 2.3:1), or PC alone lacked the capacity to inhibit urate-induced CL. However, incorporation of apoB-100 into liposomes via cholate dialysis rendered them nearly as inhibitory for urate-induced neutrophil CL as LDL on a protein weight basis. Moreover, delipidated apoB-100, containing less than 3% residual phospholipid, inhibited neutrophil responses to urate crystals or latex beads (degranulation and superoxide anion release) in a stimulus-specific manner. Modifications of the lysine residues of apoB (e.g. acetylation) reduced both the capacity of LDL to inhibit urate crystal-induced CL and to bind to urate crystals. The effects of apoB lysine residue modification were reversible, proportional to the extent of modification, and were not attributable to alteration of the net charge of apoB. Thus, the apoB-100 of LDL both mediates and shares the capacity of native LDL to inhibit certain neutrophil responses to particulates.     

Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.     

Isolation and partial characterization of genomic clones coding for a human pro-alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene

We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene.     

The effect of starvation on RNA: DNA ratios and growth of larval striped bass, Morone saxatalis

Hatchery reared larval striped bass, Morone saxatilis , 8-days-post-hatching were subjected to various feeding/starvation regimes over a period of 14 days.
Batches of larvae from each treatment were sampled over the 14-day period and subdivided for determination of notochord length and RNA:DNA ratio. The best growth was found in fully fed F1000 larvae (exposed to 1000 Artermia nauplii l⁻¹), which reached 8.2 mm after 11 days and 9.6 mm after 14 days. Starved animals after 11 days had notochord lengths of 4.9 mm. Growth curves from feeding-delayed larvae indicated that animals fed after up to 5 days starvation were capable of complete recovery. F100 larvae (exposed to 100 Anemia nauplii 1^−l) had a slower growth rate than F1000 larvae, reaching a notochord length of 7.3 mm after 14 days. RNA:DNA ratios over time closely followed notochord growth curves, with clear differences between starved, F100 and F1000 larvae being established after only 2 days. Equilibrium RNA:DNA ratios of 3.0 and 2.25 were established in F1000 and F100 larvae, respectively, 6.8 days after the beginning of the experiment. The average lag time between a change from the starved to the fed condition and a change in RNA:DNA ratio as determined by the divergence of the nucleic acid curve from the starved condition was 0.66 days.
In treatments where starvation followed various periods of feeding, larvae regressed in notochord length such that the final length at 14 days reflected the degree of feeding. RNA:DNA ratios in these animals again closely followed growth curves with a lag time of 0.81 days.
It was concluded that RNA:DNA ratios provided very accurate indices of growth in striped bass larvae which were highly sensitive to feeding status.     

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.